The present invention relates to new antifungal polypeptides that exhibit broad-spectrum antifungal activity against pathogenic and other fungi. Specifically, the present invention relates to a variant amino acid sequence defensin protein derived from an alfalfa antifungal protein and its use as an antifungal polypeptide in plant disease control against plant pathogenic fungi. The present invention relates to the antifungal polypeptides obtainable from plants in the genus Medicago. The antifungal polypeptides may be applied directly to a plant, applied to a plant in the form of microorganisms that produce the polypeptides, or the plants may be genetically modified to produce the polypeptides. The present invention also relates to microorganisms and plants transformed with DNA sequences encoding the amino acid sequence variant alfalfa antifungal protein (AFP), and compositions useful in controlling plant pathogenic fungi.
Protection of agriculturally important crops from insects and diseases has become a major concern in the agricultural industry. Fungus infection is a particular problem in damp climates and is additionally a major concern during crop storage. Plants have developed a certain degree of natural resistance to pathogenic fungi; however, modern growing methods, harvesting and storage systems frequently provide a favorable environment for plant pathogens.
Adding to the problem is the number of different fungi that may cause problems. Fungal damage can be caused by a fungus of genera such as Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Gaeumanomyces, Helminthosporium, Macrophomina, Nectria, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Puccinia, Pythium, Pyrenophora, Pyricularia, Rhizoctonia, Scerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, and Verticillium among others. Therefore, fungicidal compounds are not always effective because antifungal activity associated with a particular compound may be limited to a few species.
One approach to inhibiting plant pathogenic activity has been to identify and isolate compounds that exhibit high levels of activity against these pathogens. Several classes of polypeptides and proteins exhibiting antifungal activity against a variety of plant pathogenic fungi have been isolated (Bowles, 1990; Brears et al., 1994). The antifungal polypeptides and proteins include chitinases, cysteine-rich chitin-binding proteins, β-1,3-glucanases, permatins (including zeamatins), thionins, ribosome-inactivating proteins, and non-specific lipid transfer proteins. These proteins are believed to play important roles in plant defense against fungal infection. The use of natural protein products to control plant pathogens has been demonstrated, for example, in EPO 0 392 225.
Recently, another group of plant proteins has been found to function as defensins in combating infections by plant pathogens (PCT International Publication WO 93/05153). The plant defensins are a family of small proteins that possess potent antimicrobial and antifungal activity (for review, see Broekaert et al., 1997). The plant defensins are characterized by a conserved pattern of eight cysteine residues forming what has been referred to as a cysteine-stabilized α-helix that stabilizes folding characteristics of these proteins (Kobayashi et al., 1991). Multiple sequence comparisons of several defensins reveal that these proteins have eight cysteines, two glycines, an aromatic residue, and one acidic residue in common (Broekaert et al., 1995). This small degree of sequence conservation suggests that the cysteine-stabilized α-helix motif provides a scaffold for accommodating a variety of antimicrobial activities. Two small cysteine-rich proteins isolated from radish seed that exhibit this conserved motif, Rs-AFP1 and Rs-AFP2, were found to inhibit the growth of many pathogenic fungi when the pure protein was added to an in vitro antifungal assay medium. Transgenic tobacco plants containing the gene encoding Rs-AFP2 protein were found to be more resistant to attack by fungi than non-transformed plants. Defensin amino acid sequence variants of the radish Rs-AFP2 were identified that exhibited improved antifungal activity (De Samblanx et al, 1997). Certain amino acid modifications to non-conserved amino acids based on the alignment of the protein with a host of other plant defensins resulted in improvement in the antifungal activity of Rs-AFP2, particularly in the presence of Ca2+. The amino acid variants all contained an arginine substitution for a naturally occurring amino acid, which increased the net positive charge on the resulting amino acid sequence variant.
Proteins similar to radish seed Rs-AFP2 have been isolated from seeds of other plants (WO 93/105153; Broekaert et al., 1995). All the proteins in this group share similarity in their amino acid sequence, but differ in their antifungal activities against various fungi, especially in the presence of different mono- and divalent salts. The activity of some antifungal proteins is dramatically reduced in the presence of 1 mM CaCl2 and 50 mM KCl (Terras et al., 1992). The usefulness of an antifungal protein for genetically engineering plant disease resistance can be greatly influenced by the sensitivity of the antifungal activity to salt concentration, since metal ions such K+, Na+, Ca2+ and Mg2+ are required for normal physiological functions and are therefore abundantly present in plant cells. Furthermore, the small size of these proteins suggests that minor modifications to the amino acid sequence could effect dramatic changes in the biological activity of the proteins.
Recombinant DNA technology has recently led to the development of transgenic plants that can express proteins that have antimicrobial activity against certain pests. For example, methods for transforming a wide variety of different dicotyledonous plants and obtaining transgenic plants have been reported in the literature (see Gasser and Fraley, 1989; Fisk and Dandekar, 1993; Christou, 1994). Similarly, methods for producing transgenic plants among the monocotyledonous plants are also well documented. Successful transformation and plant regeneration have been achieved in asparagus (Asparagus officinalis; Bytebier et al. 1987), barley (Hordeum vulgare; Wan and Lemaux, 1994), maize (Zea mays; Rhodes et al. 1988; Gordon-Kamm et al., 1990; Fromm et al. 1990; Koziel et al., 1993), oats (Avena sativa; Somers et al., 1992), orchardgrass (Dactylis glomerata; Horn et al., 1988), rice (Oryza sativa; including indica and japonica varieties; Toriyama et al., 1988; Zhang et al., 1988; Luo and Wu 1988; Zhang and Wu, 1988; Christou et al., 1991), rye (Secale cereale; De la Pena et al., 1987), sorghum (Sorghum bicolor; Cassas et al., 1993), sugar cane (Saccharum spp.; Bower and Birch, 1992), tall fescue (Festuca arundinacea; Wang et al., 1992), turfgrass (Agrostis palustris; Zhong et al., 1993), and wheat (Triticum aestivum; Vasil et al., 1992; Troy Weeks et al., 1993; Becker et al., 1994).
A number of publications have discussed the use of plant and bacterial glucanases, chitinases, and lysozymes produced in transgenic plants exhibiting increased resistance to various microorganisms such as fungi (EP 0 292 435, EP 0 290 123, EP 0 392 225, EP 0 307 841, EP 0 332 104, EP 0 440 304, EP 0 418 695, EP 0 448 511, WO 91/06312, WO 88/00976, WO 90/07001 and U.S. Pat. No. 4,940,840). The protection obtained from expression of osmotin-like proteins is discussed in WO 91/18984. Alfalfa AFP (AlfAFP) is a member of the plant defensin family isolated from the seeds of alfalfa, Medicago sativa, and exhibits broad spectrum antifungal activity including activity against the potato pathogen Verticillium dahliae and the wheat pathogen Fusarium graminearum (U.S. Pat. Nos. 6,121,436, and 6,316,407). Expression of AlfAFP in potato was shown to confer resistance to early dying disease caused by Verticillium in potato (Gao et al, 2000). However, plants expressing AlfAFP also show a reduction in potato tuber size. AlfAFP was unable to provide an adequate level of protection to plants infected with Fusarium head blight even though the protein exhibited in vitro efficacy against Fusarium graminearum. 
There is thus a continuing need to identify biocidal compounds, particularly those that will be effective against plant pathogenic fungi, whether applied as compositions directly to an infected plant or expressed in transgenic plants in amounts sufficient to provide protection against the pathogens.